pH Sensing Technique Based On Graphene Electrodes

ABSTRACT

Provided are devices and methods for a rapid, non-perturbative and energy-efficient technique for pH sensing based on a flexible graphene electrode. This technique does not require the application of gate voltage or source-drain bias, and demonstrates fast pH-characterization with precision. The disclosed technology is suitable for in vivo monitoring of tumor-induced pH variation in tissues and detection of pH changes as required in a DNA sequencing system.

RELATED APPLICATION

The present application claims priority to and the benefit of U.S.patent application Ser. No. 62/308,069, “pH Sensing Technique Based OnGraphene Electrodes” (filed Mar. 14, 2016), the entirety of whichapplication is incorporated herein by reference in its entirety for anyand all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under W911NF1010093awarded by the Defense Advanced Research Projects Agency and the UnitedStates Army Research Office. The government has certain rights in theinvention.

TECHNICAL FIELD

The present disclosure relates to the field of pH measurement and to thefield of graphene devices.

BACKGROUND

Accurate and in vivo measurement of pH is vital for medical diagnosis,treatment, and health care. Tumors, for instance, tends to be moreacidic (0.5˜1.2 pH unit) than normal tissues. pH sensors based onconventional materials such as glass and silicon suffer from large size,slow response, mechanical fragility, size-limitation, etc. Accordingly,there is a need in the art for improved pH measurement methods andimproved pH measurement devices.

SUMMARY

Provided herein are graphene based pH sensors that do not require theapplication of gate voltage or source-drain bias, and demonstrate fastpH measurement so as to be suitable for in-vivo monitoring. Thesedisclosed devices avoid the hazards presented by traditional glass-basedpH meters, and also have a flexible and scalable design. New aspects ofthis design include low level signal measure, high sensitivity, being anon-FET device, being, less prone to artifacts, and having no currentflow to confound measurements.

Disclosed here is, inter alia, a technique for measuring low-levelFaradaic charge-transfer current (fA) across the graphene/solutioninterface via real-time charge monitoring of graphene microelectrodes inionic solution. This technique enables the development of flexible andtransparent pH sensors that are useful in, inter alia, in vivoapplications.

The presented pH-sensing technique, based on graphene electrodes,overcomes the shortcomings of pH sensors based on GFETs, without alsocompromising performance. Examples of the graphene electrodes are gatedthrough the electrostatic potential of the ionizable groups that adsorbon graphene. The measurement does require application of a gate-voltageor source-drain bias, so the device minimally perturbs the system underinvestigation and also consumes minimal power during its own operation.As one example, for an ionic solution with pH around 7, it only takesabout 5 seconds (or less) to achieve a precision of ˜0.1 pH units, farmore precise than what may be needed to distinguish the pH variationinduced by a tumor, for example.

In one aspect, the present disclosure provides sensor devices,comprising: a substrate layer; the substrate being at least partiallysurmounted by a conductive contact; a graphene electrode in electroniccommunication with the conductive contact; and an amount of passivationmaterial disposed atop the graphene electrode, the amount of passivationmaterial having a window formed therein so as to expose a sensingportion of the graphene electrode to the environment exterior to thesensor device.

Also provided are methods of measuring an electronic characteristic of asample, comprising: contacting the sample to the sensing portion of thegraphene electrode of a device according to the present disclosure;measuring a Faradaic current associated with contact between the sampleand the sensing portion of the graphene electrode; and estimating anelectronic characteristic of the sample based on the measured Faradaiccurrent.

Also disclosed are methods, the methods comprising estimating the pH ofa sample from a Faradaic current associated with contacting the sampleand a graphene electrode.

Additionally provided are methods of fabricating a device, comprising:disposing an amount of graphene on a substrate, the disposing beingperformed so as to place the graphene into electronic communication witha conductive contact; disposing an insulating material atop thegraphene; and defining a window in the insulating material so as toexpose at least a portion of the graphene.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 provides an exemplary workflow of the device-fabrication processand the as-fabricated flexible devices;

FIG. 2 provides an exemplary setup for characterizing the pH sensingproperties of the graphene electrode with the electrometer;

FIG. 3 provides real-time Faradaic charge transfer for various pH valuesmeasured by the electrometer (the solid lines are linear-fit);

FIG. 4 provides an exemplary dependence of Faradaic current to pHvalue—hollow symbols represent the Faradaic current measured as the pHreversed; and

FIG. 5 provides a relative steady-state Faradaic current for serum ascompared to the PBS baseline in the pH range 6.06-7.60.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms “a,”“an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. The term “plurality”, as usedherein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it can be understoodthat the particular value forms another embodiment. All ranges areinclusive and combinable.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges include each and every value within that range.

Provided here are, inter alia, devices and methods for effecting arapid, non-perturbative and energy-efficient technique for pH sensingbased on a flexible graphene electrode. This technique does not requirethe application of gate voltage or source-drain bias, and demonstratesfast pH-characterization with precision that would be suitable for invivo monitoring of tumor-induced pH variation in tissues and detectionof pH changes as required in a DNA sequencing system. The technology maybe applied to testing of biological samples (e.g., in vitro, in vivo),as the technology may be made in flexible and bio-compatible devices.

Applications of the disclosed technology include fast andultra-low-power pH sensing through small-volume samples (˜10² μL) iflarge-scale sample-preparation is not feasible. Real-time in vivomonitoring of the pH of tissues may be performed, while at the same timeavoiding the hazards that may be caused by traditional glass-based pHmeters. Real-time detecting of pH changes as part of a system for DNAsequencing may also be performed.

Existing technologies have been under development for some time butsuccessful commercialization remains a challenge. Further, a majorcomplication in existing technologies is encapsulation of sensitiveparts of the device that cannot survive contact with bodily fluids. Thisissue is essentially eliminated for graphene devices, as such devicesare biocompatible.

The present technology presents a number of advantages. First, thedisclosed graphene electrodes are flexible, safe, provide comparativelyfast readout (seconds), minimally perturbative, ultra-low power, andhave a signal that is reversible. Compared to conventional glass-basedpH meters, a graphene electrode may be relatively small, and can befabricated on a polymer substrate that is compatible with body tissueand also free of the hazards presented by broken glass. Further,compared with graphene FETs, the graphene electrode characterizes the pHthrough direct charge measurement, without the need for a swept gatevoltage or source-drain bias. This in turn makes the disclosed devicesfaster, intrinsically more energy efficient, and minimally perturbativeto the system under measurement. The disclosed graphene electrodes alsodemonstrated better reversibility than graphene FETs.

Exemplary Embodiments

Gold contacts were pre-fabricated on a piece of flexible Kaptonpolyimide substrate. An inch-size graphene sheet synthesized via ambientpressure chemical vapor deposition on copper was transferred to thesubstrate and fabricated into electrodes using photolithography andoxygen plasma etching. A 7 micrometer thick SU-8 (2007, Microchem)bio-compatible passivation layer was created to cover the gold electrodewith a 100 μm×100 μm window through which the graphene electrode isexposed.

An electrometer was used to measure charge transfer between the grapheneelectrode and ˜10² μL 150 mM NaCl aqueous solutions with various pHvalues. The Faradic current extracted from five-second ofcharge-transferring measurement decreases monotonically with respect tothe pH values, and thus may be used to characterize the pH instantly.The reversibility of the graphene electrode was also confirmed.

Graphene Sheet Preparation

Copper foil (99.8% purity) was loaded into a four-inch quartz tubefurnace and annealed for 30 minutes at 1050° C. in an ultra-high-purityhydrogen atmosphere (99.999%) with a flow rate of 80 sccm and a pressureof 850 mT at the tube outlet in order to remove oxide residues. Methaneas a chemical vapor deposition (CVD) precursor was passed in with a flowrate 45 sccm, and graphene was then deposited for 60 min.

Device Fabrication

The graphene on copper growth substrate was coated with a 500 nm layerof poly(methyl methacrylate) (PMMA, Microchem). The PMMA/graphene/coppertrilayer was then immersed in a 0.1 M NaOH solution and connected to thecathode of a power supply. With application of DC current of ˜1 A, thePMMA/graphene bilayer was peeled off from the copper substrate. Afterthoroughly rinsing with DI-water, the PMMA/graphene bilayer wastransferred onto a Kapton film with 5 nm/40 nm Cr/Au contacts that werepre-fabricated through photolithography and thermal evaporation. Afterremoval of PMMA with acetone, the film was annealed on an open-air hotplate at 200° C. for 1 hour.

A 5 nm Al₂O₃ sacrificial layer was deposited onto graphene with e-beamevaporation. Then, a 500 μm×500 μm graphene electrode was defined byphotolithography with photoresist AZ 5214E (MICROCHEM). In thedeveloping process, the Al₂O₃ sacrifice layer together with thephotoresist, except at the region that covered the graphene electrode,were removed by the organic basic developer AZ 422 MIF (MICROCHEM).Graphene that was unprotected by the Al₂O₃/photoresist was then removedby oxygen plasma etching. A passivation layer of 7 μm thick photoresistSU-8 (MICROCHEM) was created via photolithography process with a 100μm×100 μm window that exposed the graphene electrode. The photoresistand Al₂O₃ over the graphene electrode was then removed with photoresistremover 1165 (SHIPLEY) and subsequently AZ 422 MIF. At last the wholeas-fabricated film was hard-baked at 200° C. on an open-air hotplate for2 hours. The process and an as-fabricated device is shown in FIG. 1 .

Device pH Characterization

An electrometer (Keithley 5217a) with comparatively low noise was usedto measure the small scale Faradaic charge-transfer process (<pC/s) atthe graphene/solution interface. The setup of the measurement is shownin FIG. 2 . The noninverting input of the operational amplifier in theelectrometer was initially grounded.

Aliquots of aqueous NaCl solution (˜10² μpL , with an ionic strength of150 mM that is typical of bodily fluids in vivo) with various pH valueswere exposed to the graphene electrode through the 100 μm×100 μm windowof the passivation layer. When the measurement commenced, the grapheneelectrode was connected to the inverting input of the operationalamplifier. The charge that transferred from the solution to graphene andaccumulated on the feedback capacitor was indicated as the readout ofthe electrometer.

The Faradaic charge transfer between the graphene and the ionicsolutions changed linearly with time as shown in FIG. 3 . The slope ofthe linear curve is taken as the Faradaic current i. At a comparativelyhigh pH (near 11), electrons transfer from the solution to the graphene,inducing negative Faradaic current.

As the pH decreases, the Faradaic current becomes less negative, and thecurrent becomes positive at pH of about 3.0. Compared to the swept-gatemeasurement process that typically takes several minutes for GFETs todetermine the Dirac voltage, the Faradaic current was extracted within<5 seconds in this experiment. The resolution around pH=7 for this shortperiod is significantly less than 0.5 pH units, making this techniqueespecially suitable for in vivo fast monitoring of tumor-induced pHvariation. In addition, the precision is proportional to the square rootof the monitoring time, thus can be enhanced by extending themeasurement period.

The dependence of Faradaic current with pH shows an approximately linearrelationship in the pH range of 2.2-11.2 (FIG. 4 ). The sensitivity is0.12±0.01 pA/pH based on linear-fitting. For the data in FIG. 4 , the pHwas first decreased from 11 to 2, yielding the solid data points, thenincreased from 2 to 11, as shown with the open data points. Theagreement between the measurements at each pH value tested shows thatthe measurement is reversible with negligible hysteresis. (The datamarkers in FIG. 4 correspond to the data markers in FIG. 3 .)

A further exemplary embodiment of the disclosed technology is providedin FIG. 5 , which figure illustrates exemplary testing of the pHresponse of flexible graphene electrode for human serum. For suchtesting, steady-state Faradaic current may be measured about 20 secondsafter the graphene electrode is in contact with the serum. The relativeFaradaic current (FIG. 5 ), computed by subtracting the PBS baseline(see FIG. 4 herein) from the current response to the human serum, showsa plateau in the range of pH from ˜7.0 to 7.5, but decreases by about0.3 pA as pH decreases from 7.0 to 6.0.

Without being bound to any particular theory or conditions, thispH-dependent response is larger than responses associated withphysiological changes in ionic strength, and pH alleviation from 7.0 to6.0 has been observed in the interstitial fluid of human non-metastatictumor and metastatic tumor. Accordingly, the disclosed measurementmethodology is suitable for detection of tumor- or otherwise-driven pHchanges in vivo, e.g., by observing the pH of a sample of interest.

Comparison with Existing Technologies

The disclosed graphene-electrode devices are free of many disadvantagesthat have held back commercialization of conventional pH Sensors basedon conventional materials (glass and silicon), such as slow response,mechanical fragility, and size-limitation. Thus it is more suitable tobe used for in vivo applications such as tumor monitoring or monitoringof gastric pH, which is another important application. Moreover, unlikeconventional pH meters that require large amount of liquid forfunctioning, the disclosed devices may obtain readings on only smallsample volumes, e.g., 10² μL-scale of sample.

The disclosed technology is also easier to design and fabricate than thepH-sensing techniques based on GFETs due to the fact that the electrodedoes not require a gate or bias voltage. For this reason, it is alsoultra-low-power and non-perturbative to the system under measurement.The disclosed devices also demonstrate faster responsibility and betterreversibility than conventional GFET-based devices.

One may also chemically and/or biologically functionalizing the grapheneelectrode. The charge-transfer properties of graphene/solution interfacecan be modified by the functionalization, which in turn allowsadaptation of the graphene electrode to versatile applications invarious conditions, ranges, and precisions for pH sensing. Further, thepH-sensing precision that was achieved with this graphene electrode(˜0.5 pH unit) is within the range (˜1.0 pH unit) of pH variationinduced by DNA sequencing, so the disclosed methods technique can alsobe applied to DNA amplification and detection.

Exemplary Aspects

In one aspect, the present disclosure provides sensor devices,comprising: a substrate layer; the substrate being at least partiallysurmounted by a conductive contact; a graphene electrode in electroniccommunication with the conductive contact; and an amount of passivationmaterial being disposed atop the graphene electrode, the amount ofpassivation material having a window formed therein so as to expose asensing portion of the graphene electrode to the environment exterior tothe sensor device.

The substrate layer comprises a polymer, most suitably an insulatingpolymer. Polyimide (e.g., Kapton™) is considered especially suitable.Other polymers may also be used. The substrate is also suitably flexibleand also suitably biocompatible in nature. A substrate layer comprises athickness in the range of from about 1 micrometer to about 1 cm, e.g.,from about 1 micrometer, to about 500 micrometers, from about 5micrometers to about 100 micrometers, or even from about 10 micrometersto about 50 micrometers.

The graphene electrode may be of single- or multi-layered graphene;single-layer graphene is suitable. A graphene electrode may comprise across-sectional dimension in the range of from 10 micrometers to 10,000micrometers (e.g., from about 100 to about 1000 micrometers, from about200 to about 900 micrometers, from about 300 to about 800 micrometers,from about 400 to about 700 micrometers, or even from about 500 to about600 micrometers), though this is not a requirement. The graphene may bebiologically or chemically functionalized.

The passivation material may be an electrical insulator, e.g., aceramic, an oxide (e.g., metal oxide), and the like. Photoresistmaterial is also suitable for use as a passivation material. The amountof passivation material may have a thickness in the range of from about1 micrometer to about 1000 micrometers, e.g., from about 10 to about 900micrometers, from about 50 to about 500 micrometers, or even from about100 to about 300 micrometers.

Conductive contacts may be metal, e.g., gold, copper, silver, iron, andthe like. The conductive contact may also be a non-metallic conductor(e.g., graphene, carbon nanotubes), or even a semiconductor.

The window formed in the passivation material may have a cross-sectionaldimension (e.g., width, diameter, side length) in the range of fromabout 1 micrometer to about 10,000 micrometers, e.g., from about 10 toabout 1000 micrometers, from about 100 to about 900 micrometers, fromabout 200 to about 800 micrometers, from about 300 to about 700micrometers, from about 400 to about 600 micrometers, or even about 500micrometers. The window may be round, square, rectangular, oblong,polygonal, or even irregular in shape. It should be understood that adevice may include multiple graphene electrodes and multiple windowssuch that a device presents a plurality of exposed graphene electrodes.Each electrode of a given device may be individually addressable; inthis way, a device may allow for analysis of multiple samples ormultiple analyses of the same sample.

A device may include a processing train configured to measure Faradaiccharge transfer of the graphene electrode. The processing train mayinclude, e.g., an amplifier, an electrometer, and the like. One suchtrain is shown in FIG. 2 and the related description of that FIG.

Also provided are methods of measuring an electronic characteristic of asample, comprising: contacting the sample to the sensing portion of thegraphene electrode of a device according to the present disclosure;measuring a Faradaic current associated with contact between the sampleand the sensing portion of the graphene electrode; and estimating anelectronic characteristic of the sample based on the measured Faradaiccurrent. As described elsewhere herein, pH is one suitable electroniccharacteristic for these disclosed methods. The disclosure methods mayfurther include correlating the electronic characteristic to aphysiological state (e.g., a disease state, such as a cancerous state)of the subject from whom the sample was taken.

A sample being analyzed may have a volume of less than about 1000microliters, less than about 500 microliters, or even less than about200 microliters.

Additional methods are provided, these additional methods comprisingestimating the pH of a sample from a Faradaic current associated withcontacting the sample and a graphene electrode. The Faradaic current maybe based on a change in Faradaic charge transfer over time. Thedisclosure methods may further include correlating the electroniccharacteristic to a physiological state (e.g., a disease state, such asa cancerous state) of the subject from whom the sample was taken.

A sample may have a volume of less than about 1000 microliters, lessthan about 500 microliters, or even less than about 200 microliters. Thegraphene electrode may be the graphene electrode of a device accordingto the present disclosure.

Further provided are methods of fabricating a device, comprising:disposing an amount of graphene on a substrate, the disposing beingperformed so as to place the graphene into electronic communication witha conductive contact; disposing an insulating material atop thegraphene; and defining a window in the insulating material so as toexpose at least a portion of the graphene. The methods may be performedso as to give rise to a device according to the present disclosure.

It should be understood that the disclosed devices may be utilized in anex vivo fashion in which a user extracts a sample (fluid, tissue) from asubject and then contacts that sample to the device for furtheranalysis. In some embodiments, a device may be inserted into a subjectsuch that the device contacts a fluid or other tissue of interest withinthe subject.

1-24. (canceled)
 25. A sensor device, comprising: a substrate layer; anelectrode disposed on the substrate layer, the electrode comprising aconductive contact disposed on the substrate, an amount of graphenedisposed on the conductive contact, and an amount of passivationmaterial the amount of passivation material having a window formedtherein so as to expose a sensing portion of the graphene; and aprocessing train configured to measure Faradaic charge transfer betweenthe graphene of the electrode and a sample in contact with the grapheneof the electrode, the processing train determining a pH of the samplefrom the Faradaic charge transfer between the graphene of the electrodeand the sample.